Routine clinical assessments of endocrine function are currently based on the determination of hormone concentration in blood or urine. Measurement of hormone concentration by radio-immunoassay and radio-receptorassay requires relatively sophisticated and expensive equipment and involves the handling of radioactive materials. Due to the necessary use of radioactivity as well as sophisticated and expensive equipment, the radio-immunoassay cannot be performed in the physician's office or by patients themselves in "do it yourself" tests. By substituting an enzyme for a radioisotope as a marker, most of these problems can be overcome.
"Competitive protein-binding" enzyme-immunoassays for bioactive substance, e.g., hormones, such as human lutropin (hereinafter "LH"), are known (see Fukunaga, T., Rathnam, P., Landesman, R., and Saxena, B.B., Obstetrics Gynecology, 61:102 (1983) and Singh, M., Saxena B.B., and Rathnam, P., Fertility and Sterility, 41:210 (1984)). In "competitive protein-binding enzyme-immunoassays", the hormone is covalently conjugated with a marker enzyme, e.g., alkaline phosphatase, to yield an "enzyme-labeled hormone" and a hormone-specific antibody is immobilized by, for example, covalently coupling the antibody to glass beads. Increasing concentrations of unlabelled hormones are employed to compete with the "enzyme-labeled hormone" for binding with the antibody. The net amount of the antibody-bound-"enzyme-labeled hormone" is determined by measuring the enzyme concentration colorimetrically.
In "competitive protein-binding" enzyme-immunoassays, a decrease in the amount of antibody-bound-"enzyme-labeled hormone" due to an increase in the concentration of unlabelled hormone results in a proportionate disappearance of color.
"Competitive protein-binding" enzyme-immunoassys are disadvantageous when attempting to visually evaluate the results. That is, it is difficult to visually evaluate the disappearance of color and thus expensive and sophisticated equipment must be employed.
Enzyme-immunoassays which allow for the visual evaluation of an increased color due to increasing concentrations of hormone in the sample are well known and are referred to as "sandwich" enzyme-immunoassays. "Sandwich" enzyme-immunoassays have been extensively used for the determination of the concentration of various drugs and steroids in clinical diagnosis (see Korhonen, M. K. Juntenen, K. O., and Stenman, U. H., Clinical Chemistry, 26-13:1829 (1980); Ollerich, M., Journal Clinical Chemistry Clinical Biochemistry, 18:197 (1980); and Schneiter, R. S., Lindquist, P., Wong, E. T., Rubensteine, E. E., and Ullmann, E. F., Clinical Chemistry 19:821 (1973)).
In the "sandwich" enzyme-immunoassay, the hormone-specific antibody is conjugated with a marker enzyme as opposed to the hormone being conjugated to the enzyme as in a "competitive protein-binding" enzyme-immunoassay. However, the use of such a hormone specific antibody-enzyme conjugate in a "sandwich" enzyme-immunoassay does not provide satisfactory results because it is difficult to obtain a high concentration of the conjugate near the locus of the immobilized hormone-specific antibody to effect bonding without using excessively high concentrations of the antibody conjugate.
The availability of high demand specific antibodies, especially the production of monoclonal, monospecific antibodies together with the "competitive protein-binding" and "sandwich" assays to covalently link enzymes to hormones have permitted the development of enzyme-immunoassays of LH and human chorionic gonadotropin for the detection of ovulation and pregnancy respectively.
More specifically, the concentration of estradiol and LH in serum samples of peripheral blood or urine have been used for the prediction of ovulation (see Fukunaga, T., Rathnam, P., Landesman, R. and Saxena, B. B., Obstetrics Gynecology, 61:102 (1983) and Singh, M., Saxena, B. B. and Rathnam, P., Fertility and Sterility, 41:210 (1984)). The results indicate that a 50% increase in the midcycle LH surge of the mean basal level of plasma LH is one of the best hormonal indices of impending ovulation which may occur between 24 to 56 hours after the rise in LH. This has been difficult to measure in the urine by the conventional "sandwich" assay and the present invention overcomes this problem.
Due to their lack of sensitivity, immunological tests utilizing the principle of hemagglutination inhibition or latex agglutination inhibition have not permitted the detection of the midcycle pre-ovulatory LH surge. Although radio-immunoassays provide the required higher sensitivity to detect mid-cycle LH surge in the urine and blood, such assays again require expensive and sophisticated equipment.
The clinical validation of the conventional "competitive protein-binding" enzyme-immunoassay of LH in the detection of pre-ovulatory LH surge in the urine, where the LH surge was validated by the occurrence of follicular rupture within 12 to 24 hours after the surge, and substantiated with other parameters such as the thermal shift in the BBT, spinenbarkeit of the cervical mucus, hormonal levels in serum or urine and ultrasonography, has been shown in Singh, M., Saxena, B. B. and Rathnam, P., Fertility and Sterility, 41:210 (1984)). The present invention considerably improves the mode of detection of the LH.
The enzyme-immunoassay of the present invention offers the sensitivity of a radio-immunoassay while preserving the simplicity of collection of urine samples. The sensitivity of the enzyme-immunoassay of the present invention is equal to that of the radio-immunoassay and is almost 10-fold higher than the hemagglutination inhibition assay. Further, the enzyme-immunoassay of the present invention requires less than 90 minutes and the end point, as indicated by appearance of color, is easy to interpret.
When applied to the measurement of LH in the urine, the present invention provides a rapid and reliable aid to detect pre-ovulatory LH surge to predict ovulation in women. Such a measurement is extremely useful in the management of infertility and dating the time for artificial insemination and in the selection for optimum time to aspirate mature ova for in vitro fertilization as well as in aiding women in natural family planning by abstinence during the fertile period.